Amphistomatous Leaves'

Plant Physiol. (1984) 74, 47-51 0032-0889/84/74/0047/05/$0 1.00/0 Stomatal Behavior and CO2 Exchange Characteristics in Amphistomatous Leaves' Receiv...
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Plant Physiol. (1984) 74, 47-51 0032-0889/84/74/0047/05/$0 1.00/0

Stomatal Behavior and CO2 Exchange Characteristics in Amphistomatous Leaves' Received for publication September 17, 1982 and in revised form December 20, 1982

KEITH A. Morr AND JAMES W. O'LEARY* Department ofCellular and Developmental Biology, University ofArizona, Tucson, Arizona 85721 thermal conductivity for leaves (4) make significant temperature gradients across the leaf improbable in most leaves, and although The possibility that differences in stomatal conductance between upper small differences in ambient humidity may exist between adaxial and lower surfaces of amphistomatous leaves are adaptations to differ- and abaxial surfaces, under reasonably well-stirred conditions ences in CO2 exchange characteristics for the two surfaces was investi- these differences are likely to be small. However, Jones and gated. The ratio of upper to lower stomatal conductance was found to Slatyer (5) reported a higher mesophyll resistance for CO2 enterchange little in response to light and humidity for well-watered sunflower ing through the upper stomata than for the lower, and the data (Helianthus annuus L.) plants. Stressing the plants (V, = -17 bars) and of Vaclavik (12) appear to support this conclusion. These data, rewatering 1 day before gas exchange measurements reduced upper plus consideration of the anisolateral nature of the mesophyll in conductance more severely than lower in both indoor- and outdoor-grown many C3 dicotyledonous species, raise the possibility that differplants, and caused small changes in conductance ratio with light and ent CO2 exchange characteristics may exist for CO2 entering humidity. A similar pattern was found using outdoor grown sunflower through one surface or the other, caused by either differences in and cocklebur (Xanthium strumarium L.) plants. Calculated intercellular resistance to CO2 diffusion through the intercellular spaces or CO2 concentrations for upper and lower surfaces were always close to differences in photosynthetic characteristics between palisade identical for a particular set of environmental conditions for both sun- and spongy mesophyll cells. Although differences in carbon flower and cocklebur, indicating that no differences in CO2 exchange metabolism between these two types of cells have been shown characteristics exist between the two surfaces. By artificially creating a not to exist (7), differences in electron transport reactions are CO2 gradient across the leaf, the resistance to CO2 diffusion through the indicated by differences in fluorescence characteristics between mesophyll was estimated and found to be so low that despite possible upper and lower surfaces of leaves (1). nonhomogeneity of the mesophyll, differences in CO2 exchange characThe goals of this study were to clarify responses for upper and teristics for the two surfaces are unlikely. It is concluded that differences lower stomata to environmental factors, to determine if differin conductance between upper and lower stomates are not adaptations to ences in CO2 uptake characteristics exist for the two surfaces, differences in CO2 exchange characteristics. and hence decide if the observed differences in conductance are adaptive. ABSTRACT

MATERIALS AND METHODS

Sunflower (Helianthus annuus L.) and cocklebur (Xanthium strumarium L.) were grown indoors under fluorescent light banks A significant proportion of terrestrial vascular plants have with light intensity at the top of the plant maintained at approxleaves with stomata on both surfaces (termed amphistomatous), imately 350 ,E m-2 s-'. Day-night cycle was 18 and 6 h at 32 including most open field herbs and grasses and virtually all and 27°C, respectively. Plants were watered once a day with oneannual crop plants. Amphistomaty has been discussed by quarter strength modified Hoagland solution, and as necessary Parkhurst (8) and more recently by Mott et al. (6), but these with tap water. Plants were also grown outdoors in large pots for discussions have centered on the adaptive significance of am- diurnal measurements of stomatal conductance. Water stress was phistomatous leaves as opposed to hypostomatous leaves, and created by withholding water, and stressed plants were always very little has been written concerning the adaptive significance rewatered around noon on the day prior to gas exchange measof reported differences between upper and lower stomata. Upper urements. Water potential was measured with a Wescor 33T stomata are usually distinct from lower in density, conductance, Dewpoint Hygrometer using a C-51 chamber. Sunflower was and behavior. Typically, density is higher on the lower surface, used for detailed study of the response of upper and lower but due to differences in size and response to environmental stomatal conductances to environmental factors, and both cockfactors, conductance ratios are poorly related to density ratios lebur and sunflower were used to verify the response to water for the two surfaces (10). Although often reported, few studies stress for outdoor-grown plants, and for CO2 uptake experiments. have carefully quantified these differences and discussed their Photosynthesis and transpiration were determined using a gas relevance to overall gas exchange. exchange system which allowed measurements of upper and For differences in behavior between upper and lower stomata lower surfaces independently. A clamp-on type chamber with to be important in the regulation of gas exchange, there must be the leaf forming the barrier between the two chambers was used, differences in either H20 or CO2 diffusion characteristics for the and pressure was equalized in the two chambers to prevent gas two surfaces. Differences in transpiration rate per conductance flow through the leaf. Light was provided by a 300-w cool-beam are unlikely, since the process is purely physical and gradients floodlight, or for later experiments, by a 400-w metal halide and basic pathways are likely to be similar. High values of lamp. Light intensity on the adaxial leaf surface was determined with a Li-Cor model Li- 170 light meter, using the quantum 'Supported by National Science Foundation Grant DEB 8110202. sensor, and was attenuated as necessary with ordinary cheese47

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MOTT AND O'LEARY

cloth. All leaves were illuminated only from abQve. To determine stomatal responses to light and humidity, air was pumped from outside the building, dried, and rehumidified to the desired level by bubbling part of the air stream through distilled H20 and remixing. Air streams for upper and lower chambers were humidified separately to achieve the same chamber humidity for both surfaces despite differing stomatal conductances. For photosynthesis versus internal CO2 concentration experiments, different CO2 concentrations were achieved by mixing C02-free air with 2% CO2 in air. In all cases, initial CO2 concentration was measured using a Beckman model 21 5B C02 gas analyzer set in the absolute mode. CO2 depletion was measured using an ADC Series 225 Mk. II CO2 analyzer set in the differential mode. Both analyzers were calibrated against each other and against a standard gas. Water vapor loss was measured using an EG & G model 880 Dewpoint Hygrometer, and leaf temperature was measured using a fine wire thermocouple pressed to the underside of the leaf. Photosynthesis and transpiration rates and internal CO2 concentrations were calculated according to the equations given by von Caemmerer and Farquhar (I13). Conductance measurements for outdoor-grown plants were made using a Li-Cor model Li- 1600 steady-state porometer, and meteorological data such as air temperature, humidity, and light intensity were recorded to insure that experimental days were similar in these regards.

RESULTS Effects of Environmental Factors on Upper and Lower Stomatal Conductances of Sunflower. Conductances for the two surfaces and the ratio of upper conductance to lower conductance, termed conductance ratio, were quite variable among plants and even among adjacent fully matured leaves on the same plant. For well-watered sunflower plants, upper conductance generally exceeded lower conductance slightly, leading to conductance ratios ranging from 1.0 to 2.0. Both upper and lower conductances declined curvilinearly with decreasing light intensity (Fig. 1), and the two conductances behaved in parallel, leading to constant conductance ratios across a wide range of light intensities (Fig. 2a). The variation present in conductance ratio in Figure 2a is due to variation among leaves; the conductance ratio for one leaf was remarkably constant for the light intensities used. The after-effect of water stress (, = -17 bars) relieved 24 h prior to gas exchange measurements was to reduce both upper and lower conductances from the well-watered condition. Upper conductance was more severely reduced than lower (Fig. 1), indicating a differential response of the two surfaces to stress, and leading to conductance ratios less than 1.0 (Fig. 2b). If plants were allowed to recover from stress for 4 d instead of 1 d, conductance ratios were found to be similar to nonstressed plants (Fig. 2c). A slight decline in conductance ratio is seen with decreasing light intensity for plants allowed to recover for 1 or 4 d (Fig. 2, b and c), indicating that the stomatal conductances of the two surfaces were not responding in parallel to light intensity under these conditions. The deviation from parallel is not pronounced, however, and is difficult to discern from plots of stomatal conductance alone. A milder stress (o = -12 bars) relieved 24 h prior to gas exchange measurements produced extremely variable results, including some plants with conductances and conductance ratios much higher and some much lower than for well-watered plants (Fig. 2d). Perhaps fortuitously, average conductances and conductance ratios were essentially unchanged from those of wellwatered plants. Figure 3 shows the response of stomatal conductance to vapor pressure difference across the stomatal pore for well-watered and

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STOMATAL BEHAVIOR AND CO2 EXCHANGE CHARACTERISTICS

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stressed sunflower plants. Both upper and lower conductances increased as vapor pressure differences were decreased, and, as with the light response, the two conductances behaved in parallel leading to a constant conductance ratio over the range of vapor pressure differences examined. Again, average conductance ratios were slightly greater than 1.0 for well-watered plants, but stressing the plant reduced upper conductance more than lower conductance causing conductance ratios to be less than 1.0. Stomatal Conductances of Outdoor-Grown Plants. To determine if this preferential reduction of the upper conductance by stress was restricted to plants grown in the growth room, diurnal conductances of sunflower and cocklebur plants growing outdoors were measured, and conductance ratios calculated. Meteorological conditions were extremely similar for days on which conductance measurements were taken. Data for well-watered plants show that conductance ratios were 0.8 to 1.0 over most of the day for both species (Figs. 4 and 5), rather than 1.0 to 2.0 as for growth room sunflower plants. However, stressing the plants did cause low conductance ratios for the day following rewatering in both species. CO2 Uptake Characteristics for Sunflower and Cocklebur. Photosynthetic rate was determined as a function of Ci2 for both sunflower and cocklebur plants grown under the fluorescent light banks. Photosynthesis and transpiration were measured separately for the two surfaces, and a C1 value for each surface was calculated. For both cocklebur and sunflower, these two values Abbreviations: C,, internal CO2 concentration; Ca, ambient or exterCO2 concentration; ri, resistance to CO2 diffusion through the mesophyll; A, photosynthetic rate; A and A2, photosynthetic rate of upper and lower surface, respectively.

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always in close agreement for a particular set of conditions (Fig. 6), and this was true over a wide range of external CO2 concentrations despite widely differing stomatal conductances between the surfaces in some cases. By lowering Ca in one chamber (either upper or lower) until no net CO2 exchange was occurring across that surface, a situation was created in which both sets of stomata were open, yet all net CO2 exchange was occurring across only one surface. Under these conditions, Ca = C, for one surface and C, can be calculated for the other. For sunflower, these two C, values were always close to identical, and the photosynthetic rate for the leaf at that C, was the same as the photosynthetic rate for two surfaces at the same C, value (Fig. 7). These data indicate an extremely low were

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Plant Physiol. Vol. 74, 1984

respond differently to environmental factors. However, for wellwatered sunflower plants the responses to light and humidity appear parallel, as indicated by the constant conductance ratios found. In stressed plants, both 1 and 4 d following rewatering the responses are not perfectly parallel, but the differences are not large. Although differences in behavior between upper and lower E 400C stomata probably do exist, many ofthose reported are differences only in absolute conductance changes for the two surfaces, not in conductance ratio. The surface with the higher stomatal conductance must have a larger absolute change to achieve the same proportional change as the other surface and hence main0200 tain conductance ratio constant. The problem is accentuated by the practice of reporting diffusive resistances rather than conductances. For many plants, stomatal resistances are low for most physiologically important environmental conditions, and low resistance values mean small changes in absolute value for large percentage changes and hence large changes in diffusion 200 400 600 rates. The use of aspirated diffusion porometers compounds the Lower Internal (C02) ppm problem because these devices are inaccurate at low resistance FIG. 6. Internal CO2 concentration calculated for the upper surface values. Finally, although no time-course data were taken, we observed that the upper stomata generally responded more slowly versus that calculated for the lower surface under a particular set of to changes in environmental factors than did the lower, especially environmental conditions for sunflower (0) and cocklebur (0). in cocklebur. If measurements are taken before steady state is reached, differences in stomatal conductance between the two surfaces will be miscalculated. A large difference in stomatal response between upper and 40 _ lower surfaces was observed for stressed plants. Although both 'E *l * S. conductances were reduced by the treatment, the reduction in the upper conductance was much more pronounced than for the E 30_ E lower. This effect was not an artifact of the growth conditions used because it was found in outdoor-grown plants, despite 2020 differences in conductance ratios in well-watered plants for the two growth conditions. The effect was reversible within 4 d, and S ^~~St it is possible that it is the result of differing sensitivities of the < 10 _ *. two sets of stomata to ABA. Pemadasa (9) has shown that, in isolated epidermes of Commelina communis, upper stomata O I show a greater response to a given concentration of ABA than 0 400 600 do lower, and ABA levels are known to rise with water stress, Internal (C02) ppm cause stomata to close, and remain high for periods of time FIG. 7. Photosynthetic carbon assimilation versus calculated internal following resumption of high water potentials. CO2 concentration for sunflower with CO2 exchange occurrng across Any differences in CO2 uptake characteristics between the two both surfaces (0), and with CO2 exchange occurrng across only one surfaces, either due to differences in diffusional resistance to CO2 surface (*; see text). through the intercellular spaces or due to differences in photosynthetic characteristics between palisade and spongy mesophyll resistance to CO2 diffusion through the mesophyll. For cockle- cells, should be reflected in differences in calculated Ci for the bur, differences in calculated Cj for the two surfaces were found two surfaces. similarity of these two values over wide ranges under the conditions described above, with Cj for the surface of Ca and for The differences in stomatal conductance between large with no CO2 exchange across it always lower than the Cj value surfaces is strong evidence that no differences in CO2 for the other surface. Using higher Ca values accentuated these the twocharacteristics exist. This argument is supported by the differences in calculated Cj. Since there is a unidirectional gra- uptake low resistances to CO2 diffusion through the mesophyll which dient of CO2 across the leaf under these conditions, these C, our data suggest. The technique of lowering Ca for one surface values, along with the photosynthetic rate, can be used to get an until Ca = C, for that surface produces a gradient in CO2 approximation of the resistance to CO2 diffusion through the concentration across the leaf which is measurable by calculating leaf. If the gradient through the leaf is approximated as linear, the resistance to CO2 diffusion through the leaf is given by 2(Qj)! Ci for each surface. In sunflower, the resistance to CO2 diffusion A (3). Using data from cocklebur, we obtain values for this across the leaf was apparently so low that we were unable to resistance of approximately 3.0 m2 s mol~' (1.2 s cm~'). If the obtain consistently lower C, values for the surface across which maximum difference in CO2 concentration gradient within an no CO2 exchange was occurring. For cocklebur, consistently amphistomatous leaf is given by ((A A2)/(A, + A2))/(r,/2) (see differing C, values were obtained, but the calculated resistance Ref. 3), then at C, and photosynthetic rate values under ambient for CO2 diffusion across the mesophyll was only 3.0 m2 s mol-'. atmospheric conditions and high light intensities the maximum This value compares favorably with the value of 3.2 m2 s mol-' CO2 concentration difference within the leafis only 5 to 10 ppm. calculated by Farquhar and Raschke (2). However, using a technique similar to ours, Sharkey et al. (11) estimate ri in Xan:hium to be closer to 1.0 m2 s mol-', and state that they DISCUSSION believe the estimate of 3.2 m2 s mol-' to be high. Based on these It is evident from the data presented here and from various data and our measurements on sunflower and cocklebur, it seems literature sources (see 10) that upper and lower stomata often that estimated ri values are quite variable, possibly due to differE

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STOMATAL BEHAVIOR AND CO, EXCHANGE CHARACTERISTICS ences in technique, plant material, and because of the difficulty in accurately measuring the low resistances which apparently exist. Regardless of actual values, it appears that resistance across the mesophyll in many species is low, hence CO2 concentration differences within the leaf under ambient CO2 concentrations are small. Therefore, differences in resistances in the intercellular spaces or differences in photosynthetic characteristics between palisade and spongy mesophyll cells will not show up as differences in CO2 uptake characteristics between the two surfaces. In summary, differences in upper and lower stomatal response to light intensity and vapor pressure difference were found to be negligible for well-watered sunflower plants, and small for stressed ones. Stressing the plants reduced both conductances, but the effect was greater on the upper, causing a large change in conductance ratio. However, the extremely low resistances to CO2 diffusion through the mesophyll in sunflower and cocklebur preclude any differences in CO2 uptake characteristics, despite possible nonhomogeneity of the mesophyll. We conclude that differences in behavior between upper and lower stomata are not adaptations to differences in CO2 uptake characteristics for these plants. The low values for resistance to CO2 diffusion through the mesophyll found for Goss.vpium hirsutum and Zea mavs by Farquhar and Raschke (2) indicate that this conclusion may be widely applicable, but exceptions are possible for sclerophyllous or succulent leaves. LITERATURE CITED 1. BALL MC, C CRITCHLEY 1982 Photosynthetic responses to irradiance by the grey mangrove, Avicennia marina, grown under different light regimes. Plant

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Physiol 70: 1101-1106 2. FARGUHAR GD, K RASCHKE 1978 On the resistance to transpiration of the sites of evaporation within the leaf. Plant Physiol 61: 1000-1005 3. FARQUHAR, GD, S VON CAEMMERER 1982 Modelling of photosynthetic response to environmental conditions. In OL Lange, PS Nobel, CB Osmond, H Ziegler, eds, Encyclopedia of Plant Physiology, New Series. Vol 12B. Springer-Verlag, Heidelberg, pp 549-587 4. HAYES RL 1975 The thermal conductivity of leaves. Planta 125: 281-287 5. JONES HG, RO SLATYER 1972 Effects of intercellular resistance to CO2 uptake by plant leaves. Aust J Biol Sci 25: 443-453 6. MoTT KA, AC GIBSON, JW O'LEARY 1982 The adaptive significance of amphistomatous leaves. Plant Cell Environ 5: 455-460 7. OUTLAW WH, CL SCHMUCK, NE TOLBERT 1976 Photosynthetic carbon metabolism in the palisade parenchyma and spongy parenchyma of Viciafiaba L. Plant Physiol 58: 186-189 8. Parkhurst, DF 1978 The adaptive significance of stomatal occurrence on one or both surfaces of leaves. J Ecol 66: 367-383 9. PEMADASA MA 1981 Abaxial and adaxial stomatal behaviour and responses to fusicoccin on isolated epidermis of Commelina commiunis L. New Phytol 89: 373-384 10. POSPISILOVA J. J SOLAROVA 1980 Environmental and biological control of diffusive conductances of adaxial and abaxial epidermes. Photosynthetica 14: 90-127 11. SHARKEY TD, K IMAI, GD FARQUHAR. IR COWAN 1982 A direct confirmation of the standard method of estimating intercellular partial pressure of CO2. Plant Physiol 69: 657-659 12. VACLAVIK J 1974 CO2 and water vapour exchange through adaxial and abaxial surfaces of tobacco leaves of different insertion levels. Biol Plant 16: 389394 13. Von Caemmerer S. GD Farquhar 1981 Some relationships between biochemistry of photosynthesis and the gas exchange of leaves. Planta 153: 13761 387